CA2192943A1 - N-terminally extended proteins expressed in yeast - Google Patents

Abstract

The present invention relates to polypeptides expressed and processed in yeast, a DNA construct comprising a DNA sequence encoding polypeptide having the following structure signal peptide-leader peptide -X1-X2-X3-X4-X5-X6-X7-heterologous protein, X1 is Lys or Arg; X2 is Lys or Arg, X1 and X2 together defining a yeast processing site; X3 is Glu or Asp; X4 is a sequence of amino acids with the following structure (A-B)n wherein A is Glu or Asp, B is Ala, Val, Leu or Pro, and n is 0 or an integer from 1 to 5, and when n > 2 each A
and B is the same or different from the other A(s) and B(s), or X4 is a sequence of amino acids with the following structure (C)m wherein C is Glu or Asp, and m is 0 or an integer from 1 to 5; X5 is a peptide bond or is one or more amino acids which may be the same or different; X6 is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Ser, Glu, Ala and Gly; and X7 is Lys or Arg.

Description

2 1 92q43 ~ W095/35384 P~~

N-t~rm;nAlly Extended Proteins ~xpressed in Yeast FIELD OF INVENTION

The present invention relates to polypeptides expressed and processed in yeast, a DNA construct comprising a DNA sequence 5 encoding such polypeptides, vectors carrying such DNA frag-ments and yeast cells transformed with the vectors, as well as a process of producing heterologous proteins in yeast.

BACKGROUND OF THE INVENTION

Yeast organisms produce a number of proteins synthesized in-10 tracellularly, but having a function outside the cell. Such extracellular proteins are referred to as secreted proteins.
These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a presequence ensuring effective direction of the expressed product across 15 the membrane of the endoplasmic reticulum tER). The presequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation.
Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the 20 protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer, S.R. and Rothman, J.E. Ann.Rev.Biochem. 56 ~1987), 829-852).

25 Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. Euro-pean Publication No. 0088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression 30 vector harbouring DNA encoding the desired protein and a 5ignal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from WO95l3~38~ 2 1 9 2 9 4 3 PCT~K9~/00~0 -the culture medium. The signal peptide may be the desired proteins own signal peptide, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.

A problem encountered with the use of signal peptides hetero-5 logous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.

The Saccharomyces cerevisiae MF~l (~-factor) is synth~c;~d as a prepro form of 165 amino acids comprising a 19 amino 10 acids long signal- or prepeptide followed by a 64 amino acids long "leader" or propeptide, encompassing three N-linked glycosylation sites followed by ~LysArg(Asp/Glu, Ala)23~-factor)4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982), 933-943). The signal-leader part of the preproMF~l has been 15 widely employed to obtain synthesis and secretion of heterologous proteins in S. ceriYiSiae.

Use of signal/leader peptides homologous to yeast is known from i.a. US Patent No. 4,546,082, European Publications Nos.
0116201A, 0123294A, 0123544A, 0163529A, and 0123289A and 20 European Patent No. 010056lB.

In EP 0123289A utilization of the S. cerev;~iaç ~-factor pre-cursor is described whereas EP 100561 describes the utilization of the Saccharomvces cerevisiae P~05 signal and PCT Publication No. WO 95/02059 describes the utilization of 25 YAP3 signal peptide for secretion of foreign proteins.

US Patent No. 4,546,082 and European Publications Nos.
0016201A, 0123294A, 0123544A, and 0163529A describe processes by which the ~-factor signal-leader from Saccharomvces cerevisiae (MF~l or MF~2) is utilized in the secretion pro-30 cess of expressed heterologous proteins in yeast. By fusing aDNA sequence encoaing the S. cerevisiae MF~l signal/leader sequence at the 5' end of the gene for the desired protein ~ W095/35384 PCT~9S/00250 secretion and processing of the desired protein was demonstrated.

EP 206,783 discloses a system for the secretion of polypep-tides from S. cerevisiae whereby the ~-factor leader sequence 5 has been truncated to eliminate the four ~-factor peptides present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the ~-factor processing site Lys-Arg-Glu-Ala-Glu-Ala. This construction is indicated to lead to an efficient process of 10 smaller peptides (less than 50 amino acids). For the se-cretion and processing of larger polypeptides, the native ~-factor leader sequence has been truncated to leave one or two ~-factor peptides between the leader peptide and the polypep-tide.

15 A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the pep-tide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene ~Julius, D.A. et al., Cell 37 (1984b), 1075).
20 Processing of the product by the KEX 2 protease is needed for the secretion of active S. cerevisiae mating factor ~1 (MF~l or ~-factor) but is not involved in the secretion of active S. cerevisiae mating factor a.

Secretion and correct processing of a polypeptide intended to 25 be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, how-ever, the level of secretion is very low or there is no se-cretion, or the proteolytic proc~c;ng may be incorrect or 30 incomplete. As described in PCT Publication No. W0 90/10075 this is believed to be ascribable, to some extent, to an insufficient exposure of the processing site present between the C-t~rm;nAl end o~ the leader peptide and the N-t~rminAl end of the heterologous protein so as to render it Wo9s/35384 r~

in~c~cible or, at least, less ~cc~ihle to proteolytic cleavage.

Wo 9o/10075 describes a yeast expression system with improved processing of a heterologous polypeptide obtained by 5 providing certain-modifications near the processing site at the C-tprmin~l end of the leader peptide and/or the N-t~nm;nAl end of a heterologous polypeptide fused to the leader peptide. Thus, it is possible to obtain a higher yield of the correctly processed protein than is obtainable with lO unmodified leader peptide-heterologous polypeptide constructions.

SUMMARY OF THE INVENTION

The present invention describes modifications of the N-t~rm;n~l end of the heterologous polypeptide designed as 15 extensions which can be cleaved off either by naturally occurring yeast proteases before purification from the culture media or by in vitro proteolysis during or subsequently to purification of the product from the culture media.

20 The present invention relates to a DNA construct encoding a polypeptide having the following structure signal peptide-leader peptide-X1-X2-X3-X4-X5-X6-X7-heterologous protein wherein X1 is Lys or Arg;
25 x2 is Lys or Arg, X1 and x2 together defining a yeast processing site;
X3 iS Glu or Asp;
X4 iS a sequence of amino acids with the following structure (A - B) n wherein A is Glu or Asp, B is Ala, Val, Leu or Pro, and n is o or an integer Lrom 1 to 5, and when n22 ~ Wo9513s384 PCT~K95100250 each A and B is the same or different from the other A(s) and B(s), or X4 is a sequence of amino acids with the following structure (C) m wherein C is Glu or Asp, and m is O or an integer from l to 5;

X5 is a peptide bond or is one or more amino acids which may be the same or different;
lO x6 is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Glu, Ala and Gly; and X7 is Lys or Arg.

In the present context, the term "signal peptide" is under-stood to mean a presequence which is pr~dnm;n~ntly hydropho-15 bic in nature and present as an N-t~rm;n~l sequence on the precursor form of an extracellular protein expressed in yeast. The function of the signal peptide is to allow the he-terologous protein to be secreted to enter the endoplasmic reticulum. The signal peptide is normally cleaved off in the 20 course of this process. The signal peptide may be hetero-logous or homologous to the yeast organism producing the pro-tein but a more efficient cleavage of the signal peptide may be obtained when it is homologous to the yeast organism in question.

25 The expression "leader peptide" is understood to indicate a predominantly hydrophilic peptide whose function is to allow the heterologous protein to be secreted to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the medium, (i.e.
30 exportation of the expressed protein or polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the cell).

W095135384 PCT~K95100250 -The expression "heterologous protein" is intended to indicate a protein or polypeptide which is not produced by the host yeast organism in nature.

X3-X4-Xs-X6-X7 together form an extension at the N-tprm;n~l of 5 the heterologous polypeptide. This extension not only increases the fermentation yield but is due to the presence of X3, protected agains~ dipeptidyl aminopeptidase (DPAP A) proc~cc;ng, resulting in a homogenous N-t~rm;n~l of the polypeptide. The extension may be constructed in such a way lO that it will be cleaved off by naturally occurring yeast proteases other than DPAP such as, for instance, yeast aspartic protease 3 (YAP3) before purification of the heterologous protein product from the culture media.
Alternatively, the extension may be constructed in such a way 15 that it is resistant to proteolytic cleavage during fermentation so that the N-~nin~l ly extended heterologous protein product may be purified from the culture media for subse~uent in vitro maturation, e.g. by trypsin, Achromobacter lYticus protease I or enterokinase.

20 In a still further aspect, the invention relates to a process for producing a heterologous protein in yeast, comprising cultivating the transformed yeast strain in a suitable medium to obtain expression and secretion of the heterologous protein, after which the protein is isolated from the medium.

In the peptide structure (A - B) nl n is preferably 2-4 and more preferably 3.

In preferred polypeptides according to the invention X3 may be Glu, A may be Glu, B may be Ala, X5 may be a peptide bond 30 or Glu, or Glu Pro Lys Ala, or x6 may be Pro or a peptide bond.

21 92q43 ~ Wo95/35384 PCTtDK9StO02SO

Examples of possible N-t~rm;n~l extensions X3-X4-Xs-X6-X7 are:

Glu Glu Ala Glu Ala Glu ~Pro/Ala) (Glu/Lys) (Ala/Glu/Lys/Thr) Arg Ala Pro Arg, Glu Glu Ala Glu Ala Glu Pro Lys Ala (Thr/Pro) Arg, Glu Glu Ala Glu Ala Glu Ala Glu Pro Arg, Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys, Glu Glu Ala Glu Ala Glu Ala Glu Arg, Glu Glu Ala Glu Ala Glu Ala (Asp/Ala/Gly/Glu) Lys, Glu Glu Ala Glu Ala Glu Ala (Pro/Leu/Ile/Thr) Lys, Glu Glu Ala Glu Ala Glu Ala Arg, Glu Glu Ala Glu Ala Glu (Glu/Asp/Gly/Ala) Lys, Glu Glu Ala Glu Ala Pro Lys, Glu Glu Ala Pro Lys, Asp Asp Ala Asp Ala Asp Ala Asp Pro Arg, Glu Glu Glu Glu Pro Lys, Glu Glu Glu Pro Lys, Asp Asp Asp Asp Asp Lys, and Glu Glu Pro Lys.

The signal peptide se~uence of the polypeptide of the inven-20 tion ~ay be any signal peptide which ensures an effective di-W095/35384 2 9 2 9 4 3 PCT~K95/00250 -rection of the expressed polypeptide into the secretory path-way of the cell The signal peptide may be a naturally oc-curring signal peptide or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have 5 been found to be the ~-factor signal peptide, the signal peptide of mouse salivary amylase, a modified carboxypeptidase signal peptide or the yeast BAR1 signal peptide and the yeast aspartic protease 3 (YAP3) signal peptide. The mouse salivary amylase signal sequence is desc-10 ribed by 0. Hagenbuchle et al., Nature 289, 1981, pp. 643-646. The carboxypeptiaase signal sequence is described by L.A. Valls et al., Cell 48, 1987, pp. 887-897. The signal peptide is disclosed in PCT Publication No. Wo 87/02670. The yeast aspartic protease 3 signal peptide is 15 described in PCT Publication No. 95/02059.

The leader peptide sequence of the polypeptide of the inven-tion may be any leader peptide which is functional in direct-ing the expressed polypeptide through the endoplasmic reticulum and further along the secretory pathway. Possible 20 leader sequences which are suited for this purpose are natural leader peptides derived from yeast or other organisms, such as the ~-factor leader or a functional analogue thereof. The leader peptide may also be a synthetic leader peptide, e.g. one of the synthetic leader peptides 25 disclosed in PCT Publication No. W0 89/02463 or W0 92/11378.

The N-tPrm;n~lly extended heterologous protein produced by the method of the invention may be any protein which may advantageously be produced in yeast. Examples of such pro-teins are aprotinin, tissue factor pathway inhibitor or other 30 protease inhibitors, and insulin or insulin precursors, insulin analogues, insulin-like growth factor I or II, human or bovine growth hormone, interleukin, tissue plasminogen activator, glucagon, glucagon-like peptide-1 (GLP-l), Factor VII, Factor VIII, Factor XIII, platelet-derived growth fac-35 tor, enzymes, or a functional analogue of anyone of these Wogs/3s384 PCT~95/00250 gproteins. In the present context, the term "functional analogue" is meant to indicate a polypeptide with a similar --function as the native protein (this is intended to be understood as relating to the nature rather than the level of z~
5 biological activity of the native protein). The polypeptide may be structurally similar to the native protein and may be derived from the native protein by addition of one or more amino acids to either or both the C- and N-t~rm; n~l end of the native protein, substitution of one or more amino acids 10 at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
15 Such modifications are well known for several of the proteins mentioned above.

Examples of suitable insulin precursors and insulin analogues are B(1-29)-Ala-Ala-Lys-A(1-21) (as described in, e.g., EP
163 529), B(1-27)-Asp-Lys-Ala-Ala-Lys-A(1-21) (as described 20 in, e.g., PCT Publication No. 95/00550), B(1-29)-Ala-Ala-Arg-A(1-21) (as described in , e.g., PCT Publication No.
95/07931), and B(1-29)-Ser-Asp-Asp-Ala-Arg-A(1-21).

The DNA construct of the invention ~n~o~ i ng the polypeptide of the invention may be prepared synthetically by established 25 standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp. 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g.
30 in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR), e.g. as described in Sambrook et al., Molecular Clo~n;nq: A Laboratorv Manual, Cold Spring Harbor, 35 NY, 1989).

Wogs/35384 PCT~95/00250 -The DNA construct of the invent on may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide of the invention by hybridization 5 using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Clonina:
A ~aboratorv Manual Cold Spring Harbor, 1989). In this case, a genomic or cDNA sequence encoding a signal and leader peptide may be joined to a genomic or cDNA sequence encoding 10 the heterologous protein, after which the DNA sequence may be modified at a site corresponding to the amino acid sequence X1-XZ-X3-X4-Xs-X6-X7 of the polypeptide, e.g. by inserting synthetic oligonucleotides encoding the desired amino acid seauence for homologous recombination in accordance with 15 well-known procedures.

Finally, the DNA construct may be of mixed synthetic and ge-nomic, mixed synthetic and cDNA or mixed genomic and cDNA
origin prepared by annealing fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding 20 to various parts of the entire DNA construct, in accordance with standard techniques. Thus, it may be envisaged that the DNA sequence encoding the heterologous protein may be of ge-nomic or cDNA origin, while the sequence encoding the signal and leader peptide as well as the sequence encoding the N-25 t~rmin~l extension Xl-X2-X3-X4-Xs-X6-X7 may be prepared synthetically.

In a further aspect, the invention relates to a recombinant expression vector which is capable of replicating in yeast and which carries a DNA construct ~n~o~; ng the above-defined 30 polypeptide. The recombinant expression vector may be any vector which is capable of replicating in yeast organisms. In the vector, the DNA sequence encoding the polypeptide of the invention should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows 35 transcriptional activity in yeast and may be derived from ~ WO95/35384 PCT~K95/00~0 genes encoding proteins either homologous or heterologous to yeast. The promoter is preferably derived from a gene en-coding a protein homologous to yeast. Examples of suitable promoters are the Saccharomvces cerevisiae M~l, TPI, AD~ or 5 PGK promoters.

The DNA sequence encoding the polypeptide of the invention may also be operably connected to a suitable terminator, e.g.
the TPI terminator (cf. T. Alber and G. Kawasaki, J. Mol.
A~l. Genet. 1, 1982, pp. 419-434).

10 The recombinant expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in yeast. Examples of such sequences are the yeast plasmid 2~
replication genes REP 1-3 and origin of replication. The vec-tor may also comprise a selectable marker, e.g. the Schizo-15 saccharomvces pombe TPI gene as described by P.R. Russell,Gene 40, 1985, pp. 125-130.

The procedures used to ligate the DNA sequences coding for the polypeptide of the invention, the promoter and the ter-minator, respectively, and to insert them into suitable yeast 20 vectors containing the information necessary for yeast re-plication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.l. It will be under-stood that the vector may be constructed either by first pre-paring a DNA construct containing the entire DNA sequence 25 coding for the polypeptide of the invention and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, leader or heterologous protein) followed by ligation.

30 The yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, pro-duces large amounts of the heterologous protein or polypep-tide in question. Examples of suitable yeast organisms may be WO95l35384 2 1 9 2 9 4 3 PCT~K95/00250 -strains of the yeast species Saccharomvces cerevisiae, Sac-charomvces kluvveri Schizosaccharomvces ~ombe or Saccharo-mvces uvarnm. The transformation of the yeast cells may for instance be effected by protoplast formation followed by 5 transformation in a manner known ~er se. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted heterologous protein, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from 10 the medium by conventional procedures including separating the yeast cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of 15 chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.

After secretion to the culture medium, the protein may be subjected to various procedures to remove the sequence X3-X4-X5 _X6_X7 20 When x6 is Pro, Thr, Ser, Gly, or Asp, the extensions arefound to be stably attached to the heterologous protein during fermentation, protecting the N-t~rm;nAl of the heterological protein against the proteolytic activity of yeast proteases such as DPAP. The presence of an N-t~rm;nAl 25 extension on the heterologous protein may also serve as a protection of the N-t~rm;n~l amino group of the heterologous protein during ~h~m;cA1 processing of the protein, i.e. it may serve as a substitute for a BOC or similar protecting group. In such cases the amino acid qP~u~nr~ X3-X4-Xs-X6-X7 may 30 be removed from the recovered heterologous protein by means of a proteolytic enzyme which is specific for a basic amino acid (e.g. Lys or Arg) so that the t~rm;n~l extension is cleaved off at X7. Examples of such proteolytic enzymes are trypsin, Achromobac~er lvticus protease I, Enterokinase, ~ WO95/35384 2 1 9 2 9 4 3 PCT~K95100250 Fusarium ~XY~O~ l trypsin-like protease, and yeast aspartic protease 3 (YAP3) of Saccharomyces cerevisiae.

YAP3 has been characterized applying various prohormones and expressions of heterologous protein in Saccharomvces 5 cerevisiae (Niamh, X.C. et al., FEBS Letters, 332, p. 273-276, 1993; Bourbonnais, Y. et al., EMBO Journal, I2, p. 285-294, 1993; Egel-Mitani, M. et al., YEAST, 6, p. 127-137, 1990)-lO YAP3 appears during la'rge scale fermentations of zY
Saccharomvces cerevisiae. It is active when the pH is from pH

3 to about pH 6. The appearance of YAP3 is most pronounced using minimal types of media for production. In such media the cells are starved of glucose and/or nitrogen, but other 15 conditions which limit the growth conditions may be used. The YAP3 protease requires a defined motif flanking the cleavage site. Such a motif is present in the N-t~rr;nAl extension when the amino acid immediately preceding X7 iS Ala or Glu (but not when x6 is Pro, Thr, Ser, Gly or Asp).

20 Thus, when x6 is a peptide bond (and the amino acid preceding X7 iS Ala or Glu) or Ala or Glu, the extensions may to be cleaved from the heterologous protein during fermentation, most likely subsequent to the secretion thereof from the yeast cells - a process which depends on YAP3 acting on its 25 substrate (the peptide bond after lysine or arginine) in the yeast cells or in the growth media. YAP3 inside, attached to, or escaped from the yeast cells may selectively cleave off the extensions such that the non-extended product may be purified from the growth media.
30 ~he amino acid sequence X3-X7, wherein x6 Ala or Glu or is a peptide bond (and the amino acid preceding X7 iS Ala or Glu) may be removed from the heterologous protein in the medium by a process involving subjecting the yeast cells to stress to make them release YAP3 into the medium, whereby the amino 35 acid sequence X3-X7 is cleaved off.

WO 95135384 2 1 9 2 9 4 3 PcT~ss/oo~o -The stress to which the yeast cells are subjected may comprise reducing the pH of the culture medium to below 6.0, preferably below 5, or starving the yeast cells of glucose and/or nitrogen.

5 In the process of the invention for producing a heterologous protein, the yeast cell may further be transformed with one or more genes encoding a protease which is speci~ic for basic amino acid residues so that, on cultivation of the cell the gene or genes are expressed, the consequent production of 10 protease ensuring a more complete cleavage of X3 - X7 from the heterologous polypeptide.

In a preferred embodiment, one or more additional genes encoding YAP3 may be used to transform the yeast cell so that, on cultivation of the cell the gene or genes are 15 overexpressed, the consequent overproduction of YAP3 ensuring a more complete cleavage of X3-X7 from the heterologous polypeptide.

In the present context, yeast aspartic protease 3 (YAP3) embraces the native YAP3 enzyme as well as enzymes derived 20 from the native enzyme, wherein the C-t~rm;nAl has been modified in order to ensure efficient release of the YAP3 protein from the cell or wherein any modification has taken place maintaining the proteolytic activity.

Alternatively the protease may be A.lyticus protease I, 25 Enterokinase or trypsin.

The present invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed.

The invention is described with reference to the drawings, 30 wherein ~ WO9S/35384 2 1 9 2 9 4 3 PCT~K95100250 Fig. l shows a general scheme for the construction of plasmids containing genes expressing N-terminally extended polypeptides.
In Fig. l, the following symbols are used:
l: Denotes the TPI gene promoter sequence from S.
cerevisiae.
2: Denotes the region encoding a signal/leader peptide (_.g. from the ~-factor gene of S. cerevisiae).
3: Denotes the region encoding a heterologous polypeptide.
3~: Denotes the region encoding a N-t~rm;n~l extended heterologous polypeptide.

4: Denotes the TPI gene terminator sequence of S.
cerevisiae.
Pl: Denotes a synthetic oligonucleotide PCR primer det~rm;ning the structure of the N-t~rm;nAl extension.
P2~ Denotes a universal PCR primer for the amplification of region 3.
POT: Denotes TPI gene from S. ~ombe.
2~ Ori: Denotes a sequence from S. cerevisiae 2 ~ plasmid including its origin of DNA replication in S.
cerevisiae.
ApR: Sequence from pBR322 /pUCl3 including the ampicilli resistance gene and an origin of DNA replication in E. coli.

Fig. 2 shows the DNA sequence in pJB59 encoding the insulin precursor B~hnjn(l-27)-Asp-Lys-Ala-Ala-Lys-AChajn(1-21) N-t~rm;n~lly fused to the 85 residues which make up the ~-factor signal/leader peptide in which Leu in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively, and W095l35384 PCT~K95/00250 -Fig. 3 the DNA sequence of pAK623 encoding GLP-1736AI~ N-~rm;n~lly fused to the synthetic signal/leader sequence a YAP3/S 1PAVA"

Fig. 4 the DNA sequence of p~Vl42 encoding BchDjn~1-29)-Ala-Ala-Arg-Achain(1-21) N-torm;n~lly fused to the 85 residues which make up the ~-factor signal/leader peptide in which Leu in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively.

10 Fig. 5 the DNA sequence of pAK679 encoding Bchajn(1-29)-Ala-Ala-Lys-Achajn(1-21) N-t~rm;n~lly fused to the synthetic signal/leader sequence ~YAP3/LAl9~

Fig. 6 shows HPLC chromatograms of culture supernatants containing the insulin precursor Bchajn(l-27)Asp-Lys-Ala-Ala-Lys-Achajn(1-21) with or without N-t~rm;n~l extensions; and with or without in vivo or in vitro processing of the N-terminal extensions.

Fig. 7 shows the products according to figure 4 separated by size on a 10% Tricine-SDS-PAGE gel.

20 Fig. 8 shows the effect of the presence of YAP3 coexpression on the yield derived from the HPLC
data in pJBl76 compared to pJB64.

EXAMPLES

Plasmids ~nd DNA
2S All expressions plasmids are of the C-POT type (see figure l), similar to those descri~ed in WO EP 171 142, which are characterized by containing the Schizosaccharomvces Pombe triose phosphate isomerase gene (POT) for the purpose of ~ Wogs/35384 PCT~K95/00250 plasmid selection and stabilization in S. cerevisiae. The plasmids furthermore contain the S~ cerevisiae triose phosphate isomerase promoter (region l in figure 1) and terminator (region 4 in figure 1). These sequences are

5 identical to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-XbaI fragment encoding the signal/leader/product (region 2 and 3 in figure 1). In order to express different heterologous proteins, the EcoRI-~k~I =
10 fragment of pKFN1003 is simply replaced by an EcoRI-XbaI
fragment encoding the signal/leader/product of interest. Such EcoRI-XbaI fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques (cf. Sambrook et al., 1989 supra).

15 Figure 1 shows the general scheme used for the construction of plasmids containing genes expressing N-t~rm;n~lly extended polypeptides, the scheme including the following steps.

- A sample of the C-POT plasmid vector is digested with restriction nucleases EcoRV and ~kaI and the largest DNA fragment is isolated using standard molecular techniques (Sambrook J, Fritsch EL and Maniatis T, Molecular Cloning: A Laboratory Nanual, Cold Spring Harbor Laboratory Press, 1989).

- Another sample of C-POT plasmid (which may be the same or different from the plasmid mentioned above) is digested with restriction nucleases EcoRV and NcoI and the fragment comprising region l and 2 is isolated.

- Polymerase Chain Reaction (PCR) is performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main Avewalk, CT 06859, USA) according to the manufacturer's instructions and the synthetic oligonucleotide primers Pl and P2 on a template WO95/35384 PCT~K95/00250 -(which may be the same or different from the plasmids mentioned above) encoding the heterological polypeptide of interest. Pl is designed in such a way that it includes a recognition site for restriction nuclease NcoI
(5'CCATGG3') followed by the sequences encoding a ~EX2 processing site, the N-~rmin~l extension and 10-15 nucleotides identical to the sequence ~nCQ~;ng the original N-tprm;n~l of the heterologous protein of interest. P2 (e.a. 5'-AATTTATTTTAo~AAcA~TAG-3 ) is designed in such a way that it amplifies region 3 and the flanking recognition site for restriction nuclease ~I (5'-TCTAGA-3') in PCRs with Pl using standard techniques described in Sambrook et al., supra.

- The PCR product is digested with restriction nucleases NcoI and XbaI and the digested fragment is isolated.

- The fragments isolated are ligated torJether by T4 ~NA ligase under standard conditions (described in Sambrook et al., supra.

- The ligation mixture is used to transform competent E coli cells apr' and selected for ampicillin resistance according to Sambrook et al., supra.

- Plasmids are isolated from the resulting E.coli clones using standard molecular techniques (described in Sambrook et al., supra.

- DNA Sequencing is performed using enzymatic chain termination (Sequenase, United States Bi~h~iC~
according to the manufacturer's instructions in order to verify (or determine) the DNA sequences encoding the N-terminal extended polypeptide and to 21 92q43 ~ Wog5/35384 PCT~K9S/00250 ensure that it is in frame with the DNA sequence encoding the signal/leader peptide of region 2 .

- The plasmid is used to transform the yeast strain MT663 and selected for growth on glucose, as described in detail below:

Yeast transformation: S. cerevisiae strain MT663 (E2-7B XEll-36 a/~, ~tpi/~tpi, pep 4-3/pep 4-3) (the yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorglnil und Zellkulturen in connection with filing W0 92/11378 and 10 was given the deposit number DSM 6278) was grown on YPGaL (1%
Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1 lactate) to an O.D. at 600 nm of 0.6.

100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifugated and r~snsp~n~ed in 10 ml 15 of a solution containing 1.2 M sorbitol, 25 mM NazEDTA pH =
8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30~C for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM
Na2EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg 20 Novozym~234. The suspension was incubated at 30 C for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM
CaCl2, 10 mM Tris HCl (Tris =
Tris(hydluxy~ethyl)aminomethane) pH = 7.5) and r~cllqp~nd~d in 25 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 ~g of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl2, 10 mM Tris HCl, pH = 7.5) was added and the mixture left for a further 30 minutes at room 30 temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD,

6.7 mM CaC12) and incubated at 30-C for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC

W095/35384 2 1 9 2 9 4 3 PCT~K95/00250 -medium of Sherman et al., Methoc's in Yeast Genetics, Cold Spring Harbor Laboratory (1982)) containing 1.2 M sorbitol plus 2.5~ agar) at 52~C was added and the suspension poured on top of plates containing the same agar-solidified, 5 sorbitol containing medium.

Exam~le 1 Constru~tion of ~JB108 and T~JB109 Plasmid pJB59 is a derivative of pKFNlOQ3 in which the E~_RI-XbaI fragment encodes the insulin precursor Bchajntl-27)-ASP-10 Lys-Ala-Ala-Lys-Ach~jn(1-2l) N-tprminAlly fused to a signal/leader sequence corr~cip~n~;nrJ to the 85 residues of the ~-factor prepro signal peptide in which LeU in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively (Figure 2). The EcoRI-XbaI fragment is 15 synthesized in an applied biosystems DNA synthesizer (Perkin Elmer DNA Thermal Cycler) in accordance with the manufacturer's instructions.

Plasmid constructs designed to express N-tPrm;n~lly extended precursor BChojn(l-27) -ASP-Lys-Ala-Ala-Lys-A h; (1-21) 20 were obtained by means of a Pl-primer with the following sequence 5'_G~OIAIU Al~.n~ IAAGACACAAGAA~TGAAGCTGAAOCT~AC)CMAGTTCGTTMCCAACAC-3' GiyLeuSerMetAloLysArsrll~rll 'Ir~ lr~ Xaa L~ "i ~ ~'l 'iS
the P2-primer: 5'-AATTTATTTTA~TAA~ACTAG-3' and the plasmid pJB59 according to the general scheme described above. PCR
and cloning resulted in a construct wherein the DNA sequence encoding the insulin precursor Bchain(l-27)-Asp-Lys-Ala-Ala-30 Lys-Achajn(1-21) is preceded by a DNA sequence encoding the N-tPrm;n~l extension Glu-Glu-Ala-Glu-Ala-Glu-Ala-Xaa-Lys, where Xaa is either Pro (pJB108; Pro encoded by CCA) or Thr (pJB109; Thr encoded by ACA).

~ W095l3S384 PCT~K9S/00250 Example 2 Construction of PJB44, ~JB107, and ~JB126 Plasmid constructs designed to express additional N-torm;nAlly extended versions of the insulin precursor BCha~n(1-5 27)-Asp-Lys-Ala-Ala-Lys-Achajn(1-21) were made by means of a P1-primer with the following sequence Ncol S' -GGGGTATccATGGcr:A~~rAr~ArA~rrTGAAGcTGAAGcTGtcAG)(Ac)AAAGTTcGTTAAccAAcAc-3~
G~yLeuserMetAIa~ysArgGl,,r,,Al-r,,,~lrr,,Ala xoO Ly5PheVaLAsnGlnHis 10 the P2-primer: 5'-AATTTATTTTACATAACACTAG-3' and the plasmid pJB59 as described in example 1. Among the resulting plasmids pJB44, pJB107 and pJB126 where isolated. These plasmids encode theinsulinprecursorBchajn(1-27)-Asp-Lys-Ala-Ala-Lys-Achajn(1-21) preceded by the N-terminal extension Glu-Glu-Ala-Glu-Ala-Glu-15 Ala-Xaa-Lys, where Xaa is either Glu (pJB44; Glu encoded by GAA), Asp (pJB126; Asp encoded by GAC) or Gly (pJBl07; Gly encoded by GGC).

Exam~le 3.

Construction of PJB64 and ~JBllO
20 Plasmid constructs designed to express N-t~rm;nAlly extended insulin precursor Bchajn(1-27)-Asp-Lys-Ala-Ala-Lys-Achain(1-21) were obtained by means of a Pl-primer with the following sequence 25 s~GGGGTATrr-rcrrT~ C~ 'r"rGAAGCTGAAGG(AC)AAAGTTCGTTAACCAACAC-3 GlyLeuSrrMetAI~LysAr~r'~n'l~'rr'l~:lrr~ll xaa LysPheVa~AsnGlnHis the P2-primer: 5'-AATTTATTTTACATAACACTAG-3' and the plasmid pJB59 as described in example 1. Among the resulting plasmids 30 pJB64 and pJBllO were isolated. These plasmids encode N-rm;n A 1 extensions of Bchajn(l-27)-Asp-Lys-Ala-Ala-Lys-Achain(1-21) preceded by a DNA sequence encoding the N-t~rm;nAl extension Glu-Glu-Ala-Glu-Ala-Glu-Xaa-Lys, where Xaa is either Glu (pJBllO; Glu encoded by GAA) or Ala (pJB64; Ala encoded by 35 GCA).

' 2~92~43 Wo95/35384 PCT~95/00250 ExamPle 4 oonstruction of pAK663 Plasmid pAK623 is a derivative of pKNF 1003 in which the ~ç~RI-XbaI fragment encodes GLP-17 36Ala N-tPrm; nAl 1 y fused to a 5 synthetic signal leader sequence YAP3/SlpAVA (Fig. 3~.

The EcoRI-XbaI fragment was synthesized in an Applied Biosystems DNA synthesizer according to the manufacturer's instructions.

10 Plasmid constructs designed to express GLP-173~ALA with the N-tPrm;nAl extension in form of Glu-Glu-Ala-Glu-Ala-Glu-Ala-Glu-Arg was obtained by means of a P1-primer with the following sequence Ncol 15 ~-MCGTTGCCAIb4~ Ab~,u~ Ar~Tr~Tr~~~Tr~ TGcTGAAGGT-3~
AsnValAlaMetALaPrrAlaValA SlLy5A,~ al~ rgHjsA~aGl~Gly the P2-primer: 5'-AATTTATTTTAcATAA~A~TA~-3' and the plasmid pAK623 according to the method described above resulting in plasmid pAK663.

20 ExamPle 5 ExPression of N-tPr~inAl extended Products and the removal of the extensions Yeast strain MT663 transformed with the C-POT plasmids described above (pJB59, pJB108, pJB109, pJB44, pJB126, pJB107, 25 pJB64 and pJBllO) were grown on YPD (1% yeast extract, 2%
peptone and 2~ glucose) agar plates. Single colonies were used to start 5 ml liquid cultures in YPD broth pH = 6.0, which were shaken for 72 hours at 30~C. Yields of products were detPrmin~
directly on culture supernatants by the method described by 30 Snel, Damgaard and Mollerup, Chromatographia 24, 1987, pp. 329-332.

W095/35384 PCT~K95/00250 The results with the yeast strains expressing N-t~rminAlly extended Bch2jn(1-27)-Asp-Lys-Ala-Ala-Lys-Ach~in(1-21) compared to the non-extended form (pJB59) are shown below 5 Plasmid N-ter~inAl extension ~ pJB59 100%
pJB108 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Pro-Lys 275%
pJB109 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Thr-Lys 300%
pJB44 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Glu-Lys 325%*
10 pJB126 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Asp-Lys 300%
pJB107 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Gly-Lys 275%
pJB64 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys 250%*
pJBllO Glu-Glu-Ala-Glu-Ala-Glu-Glu-Lys 225%*

Yields marked by (*) asterisk denotes that the product in these 15 cases is a mixture of N-terminated extended Bchajn(1-27)-Asp-Lys-Ala-Ala-Lys-Ach~jn(1-21) andnon-extended Bcha,n(1-27)-Asp-Lys-Ala-Ala-Lys-Achajn(1-21), the latter being a result of the in vivo cleavage of the extension described below and illustrated in Figure 4a and 5.

20 In case of pAK663 expressing GLP-1736AIA N-t~rminAlly extended by Glu-Glu-Ala-Glu-Ala-Glu-Ala-Arg the yield was found to be 20 fold higher than pAK623 expressing non-extended GLP-1736AI~.

ExamPle 6 Removal of N-ter~in~Al extensions in vivo 25 Culture supernatants obtained from cultures of yeast strains transformed with plasmid pJB59, pJB64, pJB44 and pJB108 (see above) were evaluated by HPLC chromatography (Figure 4a) and parallel samples were run on a 10% Tricine-SDS-PAGE gel (Figure 5, lanes 1, 2, 3 and 4). From the HPLC chromatograms it appears 30 that the culture supernatants from yeasts with plasmid pJB44 (chromatogram 3) and pJB64 (chromatogram 2) contain both the N-t~rm;nAlly extended as well as non-extended Bchajn(1-27)Asp-Lys-Woss/3s384 PCT~K95100250 Ala-Ala-Lys-A(1-21), whereas culture supernatants from yeast with pJB108 chromatogram 4) only contain the N-~rm;r~lly extended form. In case of pJB64 about 50% of the precursor is found in non-extended form (See also Fig. 5 lane 2) whereas 5 this form only represent a minor part of pJB44.

These results illustrate the ability of yeast to cleave off N-tr~rmin~l extensions selectively in vivo when the extension is either Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys rpJB64) or Glu-Glu-Ala-Glu-Ala-Glu-AIa-Glu-Lys (pJB44) and the inability of yeast to 10 cleave off an N-terminal extension in the form of Glu-Glu-Ala-Glu-Ala-Glu-Ala-Pro-Lys (p-JB108). The proteolytic activity responsible for cleaving off the extensions may be associated with enzymes in the secretory pathway such as membrane-bound YAP3 in the ~rans-Golgi system of the yeast cells.

15 Exam~le 7 Removal of N-termin~l extensions in vitro The culture supernatants described above were used as substrates for proteolytic cleavage with either partially purified YAP3 enzyme isolated from yeast strain ME783 overex-20 pressing YAP3 (Egel-Mitani et al. 1990) or Achromobacter lvti-cus protease I.

YAP3 assay was performed as follows:
4l~1 of YAP3 enzyme 800 ~1 of cell free growth media 25 Samples were incubated for 15 h at 37~C in 0,1 M Na citrate buffer, pH 4.0 Arh~omobacter lvticus protease I assay performed as follows:
lO~g A. lYticus protease I
lml of cell free g-rowth media ~ Wogs/35384 2 1 q 2 9 4 3 PCT~K95/00250 Samples were incubated for 1 h at 37'C in 0,1 M Tris buffer, pH
8.7~

Figure 4b and 4c show the results evaluated by HPLC chro-matography obtained from the YAP3 and A. lvticus protease I
5 digestions, respectively. Parallel samples were run on 10%
Tricine-SDS-PAGE ~Fig. 5 lane 5-12).

From the chromatograms of Fig. 4b it appears that the YAP3 enzyme is able to cleave off N-terminal extensions selectively when these are in form of Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys 10 (pJB64) or Glu-Glu-Ala-Glu-Ala-Glu-Ala-Glu-Lys (pJB44) but not Glu-Glu-Ala-Glu-Ala-Glu-Ala-Pro-Lys (pJBl08) (See also Fig. 5 lane 6, 7 and 8). This results clearly indicates that YAP3 or YAP3-like enzyme(s) are responsible for the partial cleavage of N-t~rm; n~l extensions seen in vivo (cf. Example 6).

15 From the chromatograms of Fig 4c it appears that digestions with A. lvticus protease I in all cases result in the same product, namely Bchajn(1-27)-Asp-Lys-(connected by disulfide bonds)-Ach8jn(1-21) which is the end result of proteolytic cleavage after all Lys-residues found in the Bchain(1-27)Asp-Lys-20 ala-Ala-Lys-Achajn(1-21) insulin precursor including those found between the B and A chain in the precursor.

In the case of the digestion of the culture supernatant from yeast transformed with pJB59 (expressing the non-extended BchDjn(1-27)Asp-Lys-Ala-Ala-Lys-Achain(1-21)) a product is seen 25 which does not appear in the other digestions. This product, Arg-B~h8jn(1-27)-Asp-Lys-(connected by disulfide bonds)-Ach~jn(l-21), results from A. lYticus protease I cleavage of secreted leader-precursor in the growth media. A. lYticus protease I
cleaves between the Lys and Arg residues in the dibasic ~ X2 30 site of the product which has escaped KEX2 cleavage in the secretory pathway of the yeast cells.

wogs/353~ 2 ~ 9 2 9 4 3 p~ c - - -Exam~le 8 Removal of N-tennin~l extensions bv over-ex~ressina YAP3 The YAP3 gene cloned by Egel-Mitani et al. (Yeast 6 (199O) pp.
127-137) was inserted into the C-POT plasmid pJB64 encoding 5 Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys-Bchai n ( 1-27)-Pro-Lys-Ala-Ala-Lys-ACh~in(1-21) (see example 3) in the following way:

The 2.5 kb SalI/SacI fragment containing the YAP3 gene was isolated from plasmid pME768 (Egel-Mitani et al. Yeast 6 (199O) pp. 127-137) and inserted into the SalI and ~I site of lO plasmid pIC19R (March et al. Gene 32 (1984) pp. 481-485). From the resulting plasmid designated pME834 a 2.5 kb SalI/XhoI
fragment containing the YAP3 gene was isolated and inserted into the unique ~lI site placed between the POT and the Ap~
sequences of pJB64. One resulting plasmid was designated 15 pJB176.

Yeast strain MT663 was transformed with pJB176 and analyzea as described in example 5.

As can be seen from the HPLC data on yield shown in Figure 8 the presence of the YAP3 gene in pJB176 clearly effects a 20 higher percent of non-extended Bch~jn(1-27)-Pro-Lys-Ala-Ala-Arg-AChajn(1-21) in the culture-supernatant of the corresponding yeast transformants compared to the yeast transformant of pJB64.

This result illustrate the ability to make yeast strains with 25 an enhanced capacity to cleave off N-t~rm;n~l extensions selectively in vivo by manipulating the level of proteolysis caused by YAP3.

2 1 92~43 ~ WO9S/35384 PCT~K95100250 Exam~le 9 Construction of ~KVl43 Plasmid pKV142 is a derivative of pKFNl003 in which the EcoRI-XbaI fragment encodes the insulin precursor Bchajn(1-29~-Ala-Ala-5 Arg-Ach,~jn(1-21) N-t~r~in~lly fused to a signal/leader sequence corresponding to the 85 residues of the ~-factor prepro signal peptide in which Leu in position 82 and Asp in position 83 have been substituted by Net and Ala, respectively (Figure 4).

Plasmid constructs designed to express Bchajn(1-29~-Ala-Ala-Arg-10 AChajn(1-21) with a N-t~T~;n~l extension in form Asp-Asp-Ala-Asp-Ala-Asp-Ala-Asp-Pro-Arg was obtained by means of a Pl-primer with the following sequence Ncol S~-GGGGTATccATrrrTr~Acq~ aACGCTGACGCTGACCCAAGATTCGTTAACCAACACTTGTGCGG-3' GLyLeuSerMetAlaLysA,~ ,P,~,AI~.t,5'5 AsnGlnHisLeuCys the P2-primer: 5l-AATTTATTTTArA~AA~AcTAG-3~ and the plasmid pKV142 according to the general scheme described above.

Exam~le 10 20 Construction of PXVl02 Plasmid constructs designed to express Bchajn(1-29)-Ala-Ala-Arg-ACh~jn(1-21) with a N-t~rm;n~l extension in form Glu-Glu-Ala-Glu-Ala-Glu-Ala-Glu-Pro-Lys-Ala-Thr-Arg was obtained by means of a Pl-primer with the following sequence Ncol 5'~ 1Al~UAli ~r~r~r~rA~C~'rrTGAAGCTGAAGCTGAACCMAGGCTACAAGATTCGTT M CCAACACTTGTbCGb-3' bLyLeuSerMetALaLysA,L~ ~ A~ llrroLysAlaTl~ ys ~ ~r~nblnHlsLeucys the P2-primer: 5'-AATTTATTTTAC~AA~ACTAG-3' and the plasmid 30 pKVl42 according to the general scheme described above.

Wo95l3s384 2 1 9 2 9 ~ 3 r~

ExamPle 11 ExPres6ion of N-terminAl extended B~j~1-29)-Ala-Ala-Arq-A. (1-21) 5 Yeast strain ~T663 transformed with the C-POT plasmids pKV142 pKV143 and pKV102 and analyzed as described in example 5.

The results with the yeast strains expressing N-t~n;n~lly extended Bchain(1-2g)-Ala-Ala-Arg-Achain(1-21) compared to the non-extended form (pKVI42) are shown below Plasmid N-te~n;n~l extension Yi pKV142 100 pKV143:Asp-Asp-Ala-Asp-Ala-Asp-Ala-Asp-Pro-Arg 263%
pKV102:Glu-Glu-Ala-Glu-Ala-Glu-Ala-15 Glu-Pro-Lys-Ala-Thr-Arg 400 ExamPle 12 Construction ~nrl ~nression of ~IM69 and pIM70 Plasmid pAK67g is a derivative of pKFN1003 in which the EcoRI-~k~I fragment encodes the insulin precursor BchRjn(1-29)-Ala-Ala-20 Lys-Ach8jn(1-21) N-t~rm;n~lly fused to a synthetic signal/leader sequence YAP3/LA1s=(Figure S).

Plasmid constructs designed to express N-t~rm;n~lly extended insulin precursor Bchain(1-2g)-Ala-Ala-Lys-Achain(1-21) were obtained by a procedure involving two successive PCR reaction.
25 The first PCR reaction was performed by means of the primer with the following sequence 5 ~ . _ A A r A A r A A r ~ ~ -r~ -T TCr,TTAACRAACAC - 3 ' r~ rlllproL~r~ Dis ~ WO95/35384 PCT~K95/00250 the P2-primer 5'-AATTTATTTTACATAACACTAG-3' and the plasmid pAK679.

The second PCR reaction was performed by means of the Pl-primer 5Ncol S' -GTTGTTAA~ AI~I All ' ,r.~ArArAArAA 3 ValVolAsnLeulleSerMetALaLysArsGluGLu the P2-primer 5'-AATTTATTTTACATAACACTAG-3' using the PCR
10 product of the first PCR reaction as the DNA-template for the second PCR reaction.

The PCR product of the second PCR reaction was cut with NcoI
and XbaI and ligated into pAK679 according to the general scheme described above. Among the resulting plasmids two where 15 identified to encode N-t~r~inAl extensions of Bchajn(1-29)-Ala-Ala-Lys-Ach~jn(1-21) in form of Glu-Glu-Glu-Pro-Lys (pIM70) and Glu-Glu-Glu-Glu-Pro-Lys (pIM69) respectively.

Yeast strain MT663 was transformed with the C-POT plasmids pAK579, pIM69 and pIM70 and analyzed as described in example 5.

20 Whereas the yield of non-extended Bchajn(1-29)-Ala-Ala-Lys-AChajn(1-21) from yeast with pAK579 was found to be practicably nothing, yeast with pIM69 and pIM70 was found to produce large quantity of Glu-Glu-Glu-Glu-pro-Lys-Bchain(1-29)-Ala-Ala-Lys-A~hajn(1-21) and Glu-Glu-Glu-Pro-Lys-Bch~jn(1-29)-Ala-Ala-Lys-25 ACh~jn(1-21) respectively.

W 09S/35384 PCT~DK95/00250 SE~UENOE IISTING

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(iii) ANTI-SENSE: NO
(vi) oRIGINAL SOUROE :
(A) aRG~NISM: synthetic (xi) SEQUEN OE L~ lSl'lUN: SEQ ID NO: 13:
Glu Glu Pro Lys (2) lN~L__.llUN FOR SEQ ID NO: 14:
(i) SEQUEN OE ~ARA(~ .s,l~x (A) LEN3TH: 22 base pairs (B) TYPE: nucleic acid (C) STFU~ NESS: single (D) TOPOL0GY: linear (ii) MOLECULE TYPE: ~NA
(iii) ~y~~ l(~T. NO
(iii) ANII-SENSE: NO
(vi) ORIGI~LAL SOUROE:
(A) C~ r~M: synthetic (xi) SEQUEN OE L~l~ll~N: SEQ ID NO: 14:

W 095/35384 2 1 9 2 9 4 3 PCT~DK95/00250 -AA m AI m ArATAArArr AG 22 (2) lNFORMAIION ~oR SEO ID NO: 15:
(1) SEQUENOE r~ARAiH~ s~
(A) LENGTH: 63 baSe pairS
(B) TYPE: nUC1eiC aCid (C) STRANn~nN~.~.C: Sing1Q
(D) TOPOLOGY: 1inear (ii) MOLECULE TYIE: ~NA
(iii) IIY~ ~ I H ~ AT ,- NO
(iii) ANTI_SENSE: NO
(Vi) ORIGINAL SOUR OE:
(A) CR~DC3M: SYnthetiC

(X1) SEQUEN OE L~ l'lUN: SEQ ID NO: 15:
~ LAI~LA ~rr~TAAr.Ar. Ar.AArAAr~T r.AAr~--AAr. r.~NrAAArTT rrTTAArrAA 60 (2) lN~U~'l'lUN PQR SEO ID NO: 16:
(i) SE,n,~UEN OE rtlARA~ l(x (A) LENGTH: 64 baSe pairS
(B) TYPE: nuCleic aCid (C) .STRANnrrNFC.~ Sing1e (D) TOFOLOGY: 1inear (ii) MOIECUI.E TYPE: DNA
(iii) ~IY~ 11 rl~'l'l ~ 'AT~- NO
) ANII_SENSE: NO
(Vi) ORIGIN~L SOUROE:
(A) ORGANISM: SYnthetiC

(Xi) SESPEN OE L~S~hl~l'lUN: SEQ ID NO: 16:
~ W ~ LA Trr~TAAr.Ar. Ar.AArAArlrT r.AAr~rr.AAr. ~T~NNAAA~T To~TI~AOCA 60 (2) INFO~M~I~ON ~O~ SEQ I~ NO: 1~:
(i) SEQ~EN OE ~MARAIIrXISTI(X
(A~ LENGTH: 61 baSe Pa1rS
(B) TYPE: nuCleic aCid (C) ~TRANnEnN~ Sing1e ~ W O 95l35384 2 1 9 2 9 4 3 PCTADK95100250 (D) TOFOLOGY: linear (ii) MOLECUIE TYPE: ONA
(iii) ~lY~lI'hr.l I~T.: NO
(iii) ANII-SENSE: NO
(vi) CRIGINAL SoUROE:
(A) ORGANISM: synthetic (xi) SE~UEN OE L~U~lXl~lUN: SEO ID NO: 17:
~LnI~A Tr~-TAA~.A~ A~A~Ar~rT GA~ 3AAG GNAAAGTTCG TTAA~rAArA 60 (2) lN~U~ N ~oR SEO ID NO: 18:
(i) SE~UEN OE CHARA~
(A) IENGTH: 72 base pairs (B) TYPE: nucleic acid (C) ~LxANu~LN~s single (D) TOFOLOGY: linear (ii) ~OLEColE TYPE: DNA
(iii) Ily~ AL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGI~AL SoUROE:
(A) oR~ANISM: synthetic (xi) SEQUEN OE L~UXlXllON: SEO ID NO: 18:
AArrTTrrr~ TG~CTCChGC TCCAGCIAAG AGAGAI~AAG rT~AA~l-TrA Ar~-r~AAAr~A 60 rATrl~Tr.AA~. GT : 72 (2) INFORM~TION FoR SEO ID NO: 19:
(i) SE~UEN OE r~ARA~, r:~Is, I~
(A) LENGTH: 9 am m o acids (B) TYPE: am m o acid (D) TOFOILX~Y: linear ~ (ii) MOIECUIE TYPE: peptide (iii) ~y~ll~rl~l(AT.- NO
(iii) ANII-SENSE: NO
(vi) CRIGINAL SoUROE:

W 095135384 ~ 1 92 (A) oR~ANISM: synthetic (xi) SEQUE:N OE L~S~hlXll~N: SEQ ID NO: 19:
Glu Glu Ala Glu Ala Glu Ala Pro Lys l 5 (2) INFORMATION FCR SEQ ID NO: 20:
(i) SEQUEN OE ~AR~ K l .~ x (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECDLE TYPE: peptide (iii) ~Y~Jln~ AL: NO
(iii) ANII-SENSE: NO
(vi) ORIGI~AL SoUROE:
(A) aRGANISM: synthetic (xi) SEQUENOE L~S~XlXl~l~N: SEO ID NO: 20:
Glu Glu Ala Glu Ala Glu Ala Thr Lys (2) INFORM~IION FoR SEQ ID NO: 21:
(i) SEQU.EN OE ~ARA~ x l .~
(A) LENGTH: 9 amino acids (B) TY~E: amino acid (D) TOPOL~GY: linear (ii) M~LECUIE TYPE: peptide (iii) IIY~ ~1'11~'1'1~ 'AL: NO
(iii) ANTI-SENSE: NO
(vi) CRIGI~AL SoUROE :
(A) oRG~NISM: synthetic (xi) SEQUEN OE L~S~KlXl~lON: SEQ ID NO: 21:
Glu Glu Ala Glu ~la Glu Ala Glu Lys (2) lN~ l~N FoR SEQ ID NO: 22:
(i) SEQUENOE r~ARA~ X
(A) LENGIH: 9 amino acids ~ W 09S/35384 PCTADK9S1002S0 (B) TYPE: amino acid (D) TOPOLDGY: linear (ii) MOLECULE TYPE: peptide . NO
(iii) ANII-SENSE: NO
(vi) CRIGI~LAL S W R OE:
(A) ORGANISM: synl~heTic (xi) SE~UEN OE L~h~ UN: SEO ID NO: 22:
Glu Glu Ala Glu Ala Glu Ala Asp Lys (2) INFORMATION FCR SEO ID NO: 23:
(i) SE~UEN OE O~ARA~T~RT.~IICS:
(A) LENGTH: 9 amino acids (B) TYPE: am~no acid (D) TOPOLDGY: linear (ii) MOLECUIE TYPE: pepkide (iii) llY~lllL'l'l(:AL: NO
(iii) ANII-SENSE: NO
(vi) ORIGIN~L SoURCE:
(A) OR~ L3M: synthetic (xi) SE~UEN OE L~Kl~l'lUN: SEO ID NO: 23:
Glu GlU Ala Glu Ala Glu Ala Gly I~s (2) lN~L~ UN FOR SEO ID NO: 24:
(i) SE~EN OE ~AT~A~
(A) LENGTH: 8 am m o acids (B) TYPE: amino acid (D) TOPOL~GY: linear (ii) MOLEC~LE TYPE: peptide (iii) lly~uLr~ll~AL NO
(iii) ANII-SENSE: NO
(vi) ORIGINRL SOUR OE:
(A) ORGANISM: synT~helic W 095135384 2 1 9 2 9 4 3 PCT~DK95/00250 (xi) SEQUEN OE L~-XlXl'lUN: SEO ID NO: 24:
Glu Glu Ala Glu ~la Glu Ala Lys (2) lN~U~v~lUN FCR SEO ID NO: 25:
~i) SE~UEN OE C~U~RA~l~hl~
(A) LENGTH: 8 amino acids (B) ~YPE: amino acid (D) TOPOLOGY: linear (ii) MOLECUIE TYPE: peptide (iii) ~Y~ul~ l~AL: NO
(iii) ANTI-SENSE: NO
(vi) oRIGINAL SoURCE:
(A) CRGANISM: s,vnthetic (xi) SEQUEN OE L~Xl~l'lUN: SEO ID NO: 25:
Glu Glu Ala Glu Ala Glu Glu Lys (2) lN~ hJ'lUN FQR SEQ ID NO: 26:
(i) SE~UEN OE ~~ARA(~ .s~
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOFOLCX~Y: linear (ii) MO~ECULE ~YPE: peptide (iii) ~Y~ (AT~ NO
(iii) ANTI-SENSE: NO
(vi) oRIGINAL S0UROE:
(A) ORGANI5M: s,vnthetic (Xi) SEQUENOE L~XlXl'lUN: SEO ID NO: 26:
Glu Glu Ala Glu Ala Glu Ala Pro Lys (2) lN~ V~alU~ FOR SEO ID NO: 27:
(i) SE~uEN OE ~RA(~
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOL~GY: linear W o95/35384 PCTADK95/00250 (ii) MOLEC~LE TYPE: peptide (iii) ~y~!l~H~ ~T, NO
(iii) ANII-SENSE: NO
(vi) CRIGINAL SoUROE:
(A) cE~rc~M: synthetic (xi) SEQIIEN OE L~r~xlxllu~: SEQ ID NO: 27:
Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys Ala Thr Arg l 5 lO
(2) INP3RMATION PCFc SEO ID NO: 28:
(i) SEQUENOE ~ARA~T~RT~TT~C
(A) LENGTH: 8 amino acids (B) TYP~: amino acid (D) TOPOLOGY: linear (ii) MaLECUlE TYPE: peptide (iii) ~y~J,n.."~Ar.- NO
(iii) ANII-SENSE: NO
(vi) OPIGINAL SCUR OE:
(A) ORL~y~L~M synthetic (xi) SEQUEN OE LL~c~L~ cl~: SEO ID NO: 28:
Glu Glu Ala Glu Ala Glu Ala Lys (2) lN~U.~ lUN FoR SEQ ID NO: 29:
(i) SEQUEN OE C~RAC~ K1.~1 1 C ~:
(A) LENGrH: 5 amino acids (B) TYPE: amino acid (D) TcPoLaGY: line~r (ii) MaLECULE TYPE: peptide (iii) ~Y~U~ CAL: NO
(iii) ANTI-SENSE: NO
(vi) ORlGIN~L SOlIROE:
(A) aR~~DL~M synthetic (xci) SEQUEN OE LL~c~Klxllcl~: SEQ ID NO: 29:

W 095/35384 PCT~DK95/00250 Glu Glu Ala Pro Lys (2) lN~U ~_.LlU~ F~R SEO ID NO: 30:
(i) SEQUENOE r~ARAr~ l~s (A) LENGTH: 660 base pairs (B) TY1PE: nucleic aci-i (C) ~lrANL~LN~: sin31e (D) TOPOLOGY: linear (ii) M01ECUIE TYl7E: ~NA
(iii) ~iY~rllH~ l(AT~- NO
(iii) ANTI-SE~SE: NO
(vi) 0RIGINAL SOUROE:
(A) CRCANISM: synthetic (ix) FEAT~RE:
(A) NAME/KEY: CDS
(B) LOCATICN: 127..540 (ix) FEAT0PE:
(A) NAME/XEY: si~_peptide (B) LOCATION: 127..381 (ix) FEATURE:
(A) NAME/XEY: mat peptide (B) LOCATION: 382..540 (xi) SEQUENOE L~Sr~Kl~l~lU~: SEO ID NO: 30:
TAAAT~TATA ArTArAAAAA ArArATAr~-. GAATTCCATT 60 ~AArAATA~.T TCAAACAAGA Ar.ATTArAAA CIAICPAITT rATArArAAT ATAAArr.ATT 120 AAAAGA ATG AGA TIT ccr TCA AIT TrT Arr GCA GTT TrA TTC GCA GCA 168 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala TCC TCC GCA TTA GCT r~c~ CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG 216 Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr GCA CAA AIT CCG GCT GAa GCT GTC ATC GGT TAC TCA GAT TIA GAA GGG 264 Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly GAT TrC GAT GTT GCT GIT TrG CCA TIT TCC AAC AGC ACA AAT AAC GGG 312 Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly 2 ~ 92943 WO95r35384 PCT/DK95/00250 TiA TTG T~T ArA AAT ACT ACT AIT GCC AGC ATT GCT GCT AAA GAA GAA 360 Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Met Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser CAC TIG GTT GAA GCr TTG TAC TIG GIT TGC G5T GAA A5A G5T TrC TiC 456 His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe TAC ACT GAC AAG GCr GCT AAG G5T ATC GTT GAA CAA TGC TGT ACC ICC 504 Tyr Thr Asp Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser ATC TGC ICC TiG TAC CAA TIG GAA AAC TAC TGC AAC TArArrrArr 550 Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn rrr~rAr~r~rr TArAAA~TAA rATrAATATA ATTATATAAA AATATLATCT I~~ ll 610 AIA~ TG TTAT~.TAAAA TAAATTGATG ArTAfr~AAA r~TAr~rTTT 660 (2) lN~ UN FoR SEO ID NO: 31:
(i) SEÇUEN OE ~ARA(~ KI.~'1'1-~
(A) LENCIH: 138 amino acids (B) TYPE: amino acid (D) TOYOLOGY: linear (ii) MOLECULE TYP.E: protein (xi) SEQUEN OE L~Xl~l'l~N: SEQ ID NO: 31:
Met Arg Phe Pro Ser Tle Phe Ihr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Fhe Asp Val Ala Val Lsu Prs Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu - Phe Ile Asn Thr Thr Ile Ala Ser Lle Ala Ala Lys Glu Glu Gly Val Ser ~et Ala Lys Ary Phe Val Asn Gln His Leu Cys Gly Ser His Leu W O95/35384 PCTrDK95/00250 Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr ~sp Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Lle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (2) lN~U~kl'lUN FoR SEQ ID NO: 32:
(i) SEQUEN OE CH~RA~
(A) LENGTH: 516 ~ase pairs (B) TYPE: nucleic acid (C) !:~'T'I?ANrll;'.l~.'~: sirgle (D) TOPOLOGY: linear (ii) MOLECULE qYPE: DNA
(iii) ~Y~u~ ~AL: NO
(iii) ANII-SENSE: NO
(vi) CRIGINAL souROE:
(A) ORGANISM: synthetic (ix) FEAruRE:
(A) NAME/KEY: CLS
(B) LocATIoN: 133..411 (ix) FE~IURE:
(A) NAME/XEY: sigLpeptide (B) LOC~IION: 133..321 (ix) FEATURE: :
(A) NAME/REY: mat peptide (B) LOCAIION: 322..411 (xi) SEQUFN OE L~S~Xl~l'lUN: SEO m NO: 32:
T~rr~TATT ~1111~1L~U T~ X~r~r AA~TA~AAAA AA~A~ATArA GGAATTCCfi~ 60 U~rAG TICAAACAAG AAGATTP~AA ACrAICAAIT IY~ CAA TATAAA~rAr 120 ~TA~rAAAA TA ATG AAA CTG AAA ACI GTA AGA TCT GCG GTC CIT TCG 168 ~et Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser -63 ~ -60 -55 TCA crc TTT GCA TCT C~G GTC CTT GGC CAA CCA ATT GAC GAC ACr GAA 216 Ser Leu Phe Ala Ser Gln Val Leu Gly Gln Prc IIe Asp Asp Thr Glu W O95/35384 PCT~DK95/00250 TCT AAC ACT ACT TCT GTC AAC TTG AT~ GCT GAC GAC ACT GAA TCT ATC 264 Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile AAC ACT ACT TrG GTr AAC T~G GCT AAC GTT GCC AIG GCT CCA GCT CCA 312 Asn Thr Thr Leu Val Asn Leu Ala Asn Val Ala Met Ala Pro Ala Pro GCr AAG AGA CAT GCT GAA GGT ACC T~C ACC TCT GAC GTC TCG AGT TAC 360 Ala Lys Ary His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr TTG GAA G~C CAA GCT GCT A~G r~AG TTC ATC GCT TGG TlG GTr AAG GGC 408 Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly GCT TAGIY~AGAA ArTAArATTA ATATAATTAT ATAAAAATAT ~ l 461 Ala 1~1L1~1~'1~ TAGTarTAT~ TAAAATAAAT Tr~AT~AcTAc r~AAAr~TAr C m T 516 (2) lN~U._ .rl~ FCR SEO ID NO: 33:
(i) SE~UEN OE r~AR~ l W :
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUEN OE L~XlXl'l~N: SEO ID NO: 33:
Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser Gln Val L~u Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Ala Asn Val Ala Met Ala Pro Ala Pro Ala Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala l~p Leu Val Lys Gly Ala (2) lN~U.~ IlUN FCR SEO ID NO:34:
(i) SE~pEN OE ~A~A~ l.S~

W 095/35384 2 1 9 2 9 4 3 PCTrDK95100250 -~A) LENGTH: l0 amino acids (B) TYPE: amino acid (C) ~TRANnFr~.~.c: single (D) TOPOLCGY: linear (ii) MDLECULE TYPE: peptide (iii) ~Y~Ull~Ll~AL: NO
(iv) ANII-SENSE: NO
(v) FRA~MENT TYPE: internal (xi) SE~ENOE L~Xl~l~U~: SEO ID NO:34:
Asp Asp Ala Asp Ala Asp Ala Asp Pro Arg l 5 l0 (2) INFORM~IION FOR SEO ID NO:35:
(i) SE~0ENOE CHARA('I'r~
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) S~RANn~rNF-~: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: peptide (iii) HYFOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FR~MENT TYPE: internal (xi) SE~0ENOE L~ rlUN: SEO m NO:35:
Glu Glu Glu Glu Pro Lys l 5 (2) LNFORMAIION FOR SEO ID NO:36:
(i) SE 0 ENOE CHARAul~xl~
(A) LENGTH: 5 amino acids (B~ TYPE: amino acid (C) STRANDELNESS: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: peptide (iii) ~Y~Ulr~ll-~L: NO

~ W O95/35384 2 ~ 9 2 9 4 3 PCTAD~95/00250 (iv) ANTI-SENSE: NO
(v) FRA~MENT TYPE: internal (xi) SEQUEN OE L~Xl~l'lUN: SEQ ID NO:36:
Glu Glu Glu Pro Lys l 5 (2) INFORM~IION PGR SEQ ID NO:37:
(i) SEQ~EN OE CHARA~ ~Kl~
(A'l LhNGTH: 6 amino acids (B TYPE: amino acid (C, ~ N~ N~''i.';: single (D, ICPOLOGY: linear (ii) MOLECULE IYPE: peptide (iii) ~Y~J~ AL: NO
(iv) ANTq-SENSE: NO
(v) FRAaMENr TYPE: internal (xi) SEQUEN OE L~Kl~l'lUN: SEQ ID NO:37:
~sp Asp Asp Asp Asp Lys l 5 (2) INFoRM~IION FCR SEQ ID NO:38:
(i) SEQUEN OE CHARA( Irxl.~
(A) LENGTff: 61 amino acids (B) TYPE: amino acid (C) sTI~n~E~NEss: single (D) TCFoLoGY: linear (ii) MOLECUIE TYPE: peptide (iii) ~y~Ul'H~.'ll~AL: NO
(iv) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal Wo 95/3S384 2 1 9 2 9 4 3 F~ ~

(xi) SE~UENOE u~i~KL~~ SEO ID NO:38:
Glu Glu Ala Glu ~la Glu Ala Lys l~e Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu ffl Leu Val Cys Gly Glu Arg Gly Phe Fhe Tyr ~hr Fro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys ~hr Ser Ile Cys Ser Leu Tyr Gln Lsu Glu Asn Tyr Cys Asn (2) lNlUl~L~A'l'l~ F~R SEO ID NO:39:
(i) SE~ENOE CEl~A''l'~XlXl IrX
(A) LENGIH: 53 am~no acids (;3) TY~: amino a--id (C) STR~NDEDNESS: single (D) TOFOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) IIY~ ~ ~T, NO
(iv) ANTI-SENSE: NO
(v) ~A~T ~PE: internal (xi) SE~UENOE L~r~lloN: SEO ID NO:39:
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Fhe Phe Iyr Thr ~ro Lys Ala Ala Arg Gly Ile Val GlU Gln Cys Cys Thr Ser Ile Cys Ser Ieu Tyr Gln LeU
35 ~0 45 Glu Asn Tyr Cys Asn (2) INF~lICN ~ SEO ID NO:40:
(i) SEQI~ENOE ~RARAr~ xl.~
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) '~RANI~ : single (D) TOPQ~: linear (ii) MOLEC[~E TY~E: peptide 2 ~ 92943 (iii) ~lY~ 'l'L~ NO
(iv) ANTI-SENSE: NO
(v) E~T TYPE: internal (xi) SE~tlENOE ll~;~'Xl~l'LUN: SEO ID NO:40:
Asp Asp Ala Asp Ala Asp Ala Asp Pro Arg (2) INF~ION E~R SEQ ID NO:41:
(i) SEQ~ENOE rFlARA~ s~
(A) L~: 74 base pairs (B) TYPE: nucleic acid (C) SIRANTlT'i~F~C: single (D) TOPOla~Y: linear (ii) M~LEa)lE T~PE: DNA
(iii) HY~ AL: NO
(iv) ANTI-SENSE: NO
(v) E~T l'YPE: interr~al (xi) SE~13ENOE ~r~lLUN: SEQ ID NO:41:
r3~LHl~ Trr~AAr.Ar~ Ar.A~r.A~r~T r.Anr~rr.Ar~7 rTr~A~rrAAr~ ATrr~TTAA~ 60 rAArAt~lTl~.T GCGG 74 (2) IN~lION ~IR SEQ ID NO:42:
(i) SE~ENCE t'MARAt~l~F'RTSTTI~.
(A) LENGl:~: 13 amino acids (B) TYPE: amino acid (C) STRANIl ~ F~ single (D) ICPOIDGY: linear (ii) I~IECULE TYPE: Eeptide (iii) HY~ AT.- NO
(iv) ANll-SENSE: NO
(v) FRA~T TYPE: internal W ogs/35384 2 1 9 2 ~ 4 3 (xi) SEQUENOE L~lXl~lUN: SEO ID NO:42:
Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys Ala m r Arg (2) lN~L~ N FCR SEQ ID NO:43:
(i) SEQUEN OE rR~R~r~RT.s~rTrs (A) LENGT~: 83 base pairs (B) TYPE: nucleic acid (C) sT~Nn~r~F~s: single (D) IOPOLOGY: linear (ii) MDLECULE TY~E: DNA
(iii) HY~i~T('~T,- NO
(iv) AN51-SENSE: NO
(v) FR~GMENT TYPE: intern21 (xi) SEÇDEN OE ~UXl~llUN: SEO ID NO:43:
~b~L~l~A T~ T~Ar.~. Ar.~ Ar~'T ~.~Ar~nr~A~. CIGAACCAAA ~'T~hA~.~ 60 TTc~TTAArr AACASlIGTG CGG 83 (2) lN~Ur~Pl'lJ~ FOR SEO ID NO:44:
~i) SEQUEN OE rR~RA~ l s ~
(A) LENGIH: 53 aTLLno acids (B) TYPE: amino acid (C) STRANDEDNESS: s mgle (D) TO~OLOGY: linear (ii) MDLECUIE TYPE: peptide (iii) ~y~l~H~ AT.~ NO
(iv) ANII-SENSE: NO
(v) FRA3MENT TY~E: internal (xi) SE~UENOE ~LKl~lluN: SEO ID NO:44:
Phe Val Asn Gln His Leu Cys Gly Ser ~is L~u Val Glu Ala Leu Iyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys ~ W O95l35384 PCTADK95100250 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Iyr Cys Asn (2) INFoRM~IION FCR SEO ID NO:45:
(i) SEQUEN OE ~RARA~I~x~
(Al LENGTH: 39 base pairs (Bl TYFE: nucleic acid (C cTRANnFr~c single (D, TOFOLOGY: linear (ii) MDLECULE TYPE: DNA
(iii) ~Y~vln~ll~AL: NO
(iv) ANTI-SENSE: NO
(v) FFI~ iT TYPE: intern21 (xi) SEgpEN OE L~Kl~l'lUN: SEO ID NO:45:
.AA~AA~AAr. AA~.AA~AA~C AAl~rrC3IT AA~rAArA~ 39 (2) LN~ .ll~N F~R SEO ID NO:46:
(i) SE~UEN OE ~ARA(~
(A) L~NGTH: 36 base pairs (B) TYPE: nucleic acid (C) .'.I.~ANI)~:lIN~i.'i: single (D) TOPOLOGY: linear (ii) MDLECULE TYPE: DNA
(iii) ~Y~Ul~h'l'lCAL: NO
(iv) ANTI-SENSE: NO
(V) F~ DFNT TYPE: internal (xi) SE~UEN OE L~CKl~l'lUN: SEO ID NO:46:
~T~rTTAArT TGarCTCCAT ~rl~rAA~.A~.A GAhGAA 36 (2) INFORM~IION FoR SEO ID NO:47:
(i) SE~UEN OE r~ARA~ l S l l(~
(A) LENCTH: 59 amino acids (B) TYFE: amino acid (C) ~ 1 ]N~'i!; single Wo 95/3538~ ? 1 9 2 ~ ~ ~ PCT/DK95/00250 (D) TOPOLOGY: linear (ii) MOLEa~[E TS~PE: peptide (iii) llY~15~'1'1C AL: NO
(iv) ANTI-SENSE: NO
(v) E~T TYPE: internal (xi) SE~ENOE ll~iLt~l~l'lUN: SEO ID NO:47:
Glu Glu Glu Glu ~ro Lys Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr mr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (2) lN~U~VIA'L'lON F~R SEO ID NO:48:
(i) SEQUENOE ~TARA(~I~TSTT(~
(A) LENGTH: 5~ amino acids (B) TYPE: amino acid (C) ~C~?AN~.~.~: single (D) rcpoL~y: lin~3ar (ii) MoLEC~E TYPE: peptide (iii) ~iY~ AL: NO
(iv) AN'll-SENSE: N~
(v) F~r TYPE: internal (xi) SEQUENOE I~L'~ l'lUN: SEO ID NO:48:
Glu Glu Glu Pro Lys Phe Val Asn Gln His Leu q,S Gly Ser His Leu Val Glu Ala Leu ~yr Leu Val Cys Gly Glu Arg Gly Phe Phe I~r Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys ~ W 09~/35384 PCT/DK95/002~0 Ser Leu Iyr Gln Leu Glu Asn Tyr Cys Asn (2) lN~U~VL~ UN FoR SEO ID NO:49:
(i) SEÇUEN OE CHARArrFRT.CTTr.~:
- (A) LENGTff: 10 amuno acids (B) TYPE: amlno acid (C) sTRANnFrNF~c~: single (D) IOPOIDGY: linear (ii) MOLECULE TYPE: peptide (iii) ~y~ AT.- NO
(iv) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE ~Xl~l'lUN: SEO ID NO:49:
Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys (2) INFCRM~IION PoR SEQ ID NO:50:
(i) SE~UEN OE CHARA(Ir~ls~
(A) LFNGTH: 660 base pairs (B) TYPE: nucleic acid (C) STRAN~EDNESS: single (D) T5POILGY: linear (ii) ~OLECUIE TYPE: DNA
(iii) ~y~lH~ll(AT.- NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) sEguEN OE L~S~KlXl'lUN: SEO ID NO:50:
GTlTGTATTC ~ c~ K~rA AfTArAAAAA ArArATArAr. r.AATrrrATT 60 rAArAATArT TrAAArAArA A~ATTArAAA CIPlY~Frr rATArArAAT ATAAAr~ATT 120 AAAArAAT.-A ~ l~rl~ AATTTTTArT rrAr~TTTAT I~X~2~iCArC Cl~L~hll~ 180 GCIGCTCCAG TCAAC~CL~C AArA~AAr.AT r~AAArrrlrAr AAl~r~yo3Gc Tr.AArl~n~.Tr 240 A~YX~F~T rAr~ATTTArA A~c~ rrc r~A~GrrGcTG ~ rrATT TICCAAriUGC 300 2 ~ 92~43 , ~
WO 95/35384 P~,JIL _ ArAAATAArr.~ TATAAATArr ArrATT~rrA ~ L~ TAAAr.AAr.AA 360 ~ l'A rrrx~rAArAr ATrCGrrhAC rAArArrT~r ~u~r~ A rT'Tr~rTrAA 420 r~rrr~rArr ~ . T~~AAAr~Ar~T IICIIC~ACA cTccraA5GC Tr~rrAr~Ar~T 480 A~ XX~AAC AA~Y3crGTAc CTCC~ICI~C TCCITGTACC A,A~ iiAA rrArT~rAAr 540 TAr-ArrrArr OOGCAGGCTC TAr~AAArTAA r~ATTAATATA ATTATATAhA AATATTAT~T 600 1~L111~11~1 ATATCTAGTG TTAT~TAAAA TAA~FrGATr ArTArrrAAA rl~rAr~rr~T 660 (2) INFCRM~ION FOR SEQ ID NO:51:
(i) SE~UENOE r~ARA(~l~rKl~l~l(~
(A) ~ENr,TH: 600 base pairs (B) TYPE: nucleic acid (C) .'; I ~ANI 1~:1 IN~ single (D) TOFoLsGY: linear ~ OLE 01E TYPE: DNA
(1ii) 11Y1V11i~'1'1(:AL: NO
(iv) ANTI-SENSE: NO
(V) ERAGMENT TYPE: inT~Enn3l (xi) SEQUEN OE L~ l'lUl~: SEQ ID NO:51:
~ .lATr ~1~ ~ TT~AATCTAT AArrArAAAA AArArATArA G~AATTOCAT 60 TCAAGAATAG IIX~AACAAG AAr.A~TArAA ACIAICAAIr TCATACACAA TATAAArr.Ar 120 r~rArrAAAA TAATr~AAArT rAAAArTrrA AGA~K~nGcGG l~~ c ACTCTTTGCA 180 TCTCAGGTCC TDGGccaacc AATT~.Arr.Ar ACIX~AATCTA ArArrArrTc l~rrAArT~Tr~ 240 AITr~rl-T~Arr. ACACT~A~ATC Il~rK~Cr ArrAArArTA (~ 11 N'l'l' ~ .1 1 300 AACTTGATCT rrATrryrrAA GA~IICGIT AArrAArArT 1~1~L~11~ CCACLIa3TT 360 r.AArl~rr~r Arrrr~Trr~ rrl~nr.AAArA ~ ArArrrrrAA r~cnGcTAAG 420 GG~TTGTCG AArAATr~Tr. TAccIy~ATc 'l~L'l~'l'll~'l' ArrAATTr~.A AAArTArTCr 480AArrAr~Arr~r Ar~rrrrrArr~ rrrrAr~AAAr TAAr~ATrAAT ATAATTATAT AAAAATATTA 540 ~C11~ TTIaIAICTA (.I.~" ~T..I~A AAATAAATTG AT..ArTArrr. AAAr~TArrr 600 (2) ~E~ON FOR SEQ ID NO:52:

W O95/35384 PCTrD~95/00250 (i) SEQ~EN OE ~ARA~
(A) LEN3TH: 55 amono acids (B) TYPE: am m o acid (C) STRANDELNESS: single (D) TOPOLOGY: linear (ii) MOIECULE TYPE: peptide (iii) ~Y~u~h~ ~AL: NO
(iv) ANIl-SEN ~: NO
(v) FRhaMENr T~FE: internal (xi) ~ ~UENOE L~ Sl~lU~: SEQ ID NO:52:
Phe Val Asn Gln His leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ser Asp Asp Ala Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Ieu Glu Asn Tyr Cy5 Asn

Claims (22)

1. A DNA construct encoding polypeptide having the following structure signal peptide-leader peptide-X1-X2-X3-X4-X5-X6-X7-heterologous protein, wherein the heterologous protein is selected from the group consisting of aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin-like growth factor I or II, human or bovine growth hormone, inter-leukin, tissue plasminogen activator, glucagon, glucagon-like peptide-1, Factor VII, Factor VIII, Factor XIII, platelet-derived growth factor, enzymes, insulin or an insulin precursor, and a functional analogue of any of these proteins, and X1 is Lys or Arg;
X2 is Lys or Arg, X1 and x2 together defining a yeast processing site;
X3 is Glu or Asp X4 is a sequence of amino acids with the following structure (A - B)n wherein A is Glu or Asp, B is Ala, Val, Leu or Pro, and n is 0 or an integer from 1 to 5, and when n>2 each A and B is the same or different from the other A(s) and B(s), or X4 is a sequence of amino acids with the following structure (C)m wherein C is Glu or Asp, and m is 0 or an integer from 1 to 5;
X5 is a peptide bond or is one or more amino acids which may be the same or different;

X6 is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Ser, Glu, Ala and Gly; and X7 is Lys or Arg, with the proviso that the DNA construct does not encode a polypeptide comprising a fusion of a signal peptide, a leader peptide and a heterologous protein or polypeptide, which polypeptide is modified in its amino acid sequence adjacent to a yeast processing site positioned between the C-terminal end of the leader peptide and the N-terminal end of the heterologous protein so as to provide a presentation of the processing site which makes it accessible to proteolytic cleavage, the polypeptide having the following structure signal peptide-leader peptide-X1-X2-X3-X4-heterologous protein wherein X1 is a peptide bond or represents one or more amino acids which may be the same or different, X2 and X3 are the same or different and represent a basic amino acid selected from the group consisting of Lys and Arg, X2 and X3 together defining a yeast processing site, and X4 is a peptide bond or represents one or more amino acids which may be the same or different, with the proviso that X1 and/or X4 represent one or more amino acids and that at least one of the amino acids represented by X1 and/or X4 is a negatively charged amino acid selected from the group consisting of Glu and Asp.

2. A DNA construct according to claim 1, wherein X3 is Glu.

3. A DNA construct according to claim 1, wherein A is Glu.

4. A DNA construct according to claim 1, wherein B is Ala

5. A DNA construct according to claim 1, wherein n is preferably 2-4, more preferably 3.

8. A DNA constuct according to claim 1, wherein X5 is a peptide bond, Glu, Asp or Glu-Pro-Lys-Ala.

7. A DNA constuct according to claim 1, wherein X6 is Pro, or a peptide bond.

8. A DNA construct according to claim 1, wherein the signal peptide is the .alpha.-factor signal peptide, the yeast aspartic protease 3 signal peptide, of mouse salivary amylase, the carboxypeptidase signal peptide, or the yeast BAR1 signal peptide.

9. A DNA construct according to claim 1, wherein the leader peptide is a natural leader peptide such as the .alpha.-factor leader peptide, or a syntheticleader peptide.

10. A DNA constuct according to any one of the preceding claims, wherein the heterologous protein is insulin or an insulin precursor or a functional analogue thereof.

11. A DNA constuct according to any of claims 1-10, wherein the amino acid sequence X3-X7 is Glu Glu Ala Glu Ala Glu Ala Pro Lys, Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys Ala Thr Arg, Glu Glu Ala Glu Ala Glu Ala Lys, Glu Glu Ala Pro Lys, Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys, Asp Asp Ala Asp Ala Asp Ala Asp Pro Arg, Glu Glu Glu Glu Pro Lys, Glu Glu Glu Pro Lys or Asp Asp Asp Asp Asp Lys.

12. A recombinant expression vector which is capable of replicating in yeast and which carries a DNA construct according to claim 11.

13. A yeast strain which is capable of expressing a heterologous protein and which is transformed with a vector according to claim 12.

14. A process for producing a heterologous protein, comprising cultivating a yeast cell according to claim 13 in a suitable medium to obtain expression and secretion of the heterologous protein, after which the protein is isolated from the culture medium.

15. A process according to claim 14, wherein the amino acid sequence X3-X7 is removed from the recovered heterologous protein by a process involving treatment with a proteolytic enzyme which is specific for a basic amino acid.

16. A process according to claim 15, wherein the proteolytic enzyme is selected from the group consisting of trypsin, Achromobacter lyticus protease 1, Enterokinase, Fusarium oxysporum trypsin-like protease, and YAP3.

17. A process according to claim 15, wherein the amino acid sequence X3-X7, wherein X6 is a peptide bond, Ala, or Glu, is removed from the heterologous protein in the medium by a process involving subjecting the yeast cells to stress to make them release yeast aspartic protease-3 into the medium, whereby the amino acid sequence X3-X7 is cleaved off.

18. A process according to claim 17, wherein the stress to which the yeast cell are subjected comprises reducing the pH of the culture medium to below 6.0, or starving the yeast cells by subjecting them to limiting growth conditions.

19. A process according to claim 18, wherein the pH of the culture medium is reduced to below 5.

20. A process according to claim 14, wherein the yeast cell is further transformed with one or more genes encoding a protease which is specific for basic amino acid residues so that, on cultivation of the cell the gene or genes are expressed, the consequent production of protease ensuring a more complete cleavage of X3 - X7 from the heterologous polypeptide.

21. A process according to claim 20, wherein the gene coding for the protease is a gene coding for YAP3 so that, on cultivation of the cell the YAP3 gene or genes are overexpressed, the consequent overproduction of YAP3 ensuring a more complete cleavage of X3-X7 from the heterologous polypeptide.

22. A process according to claim 20, wherein the gene coding for the protease codes for A.lyticus protease I or trypsin.